The field of the invention is gene therapy, particularly the use of enveloped vectors for gene delivery.
Infection of a host cell by an enveloped virus is initiated by binding of at least one viral envelope protein to a cognate virus receptor molecule on the cell surface. The viral envelope protein not only binds to the receptor but also catalyzes fusion of the viral envelope and the host cell membrane. The presence or absence on a cell of a cognate virus receptor molecule is a primary determinant of the host range and the tissue tropism of any given virus.
Hemagglutinin (HA) is the major surface protein of influenza A virus, and it is perhaps the best-characterized membrane protein. HA is synthesized as a single polypeptide precursor, HO, which is proteolytically cleaved into two subunits HA1 and HA2, either in the late Golgi or extracellularly, depending on the nature of the cleavage site as reviewed in Klenk and Garten (Trends Microbiol. 2:39-43). HA initiates infection by binding a sialic acid-containing virus receptor molecule on the surface of a target cell (Paulson, 1985, In: The Receptors, Vol. 2, pp.131-219, Conn, ed., Academic Press, Orlando, Fla.). Detailed structural studies further revealed that there is a region in the HA1 subunit that binds sialic acid which region has been named the receptor binding pocket (RBP) (Weis et al., 1988, 333:426-431). The RBP comprises several highly conserved amino acid residues, all of which are involved in the hydrogen-bond network which defines the RBP topography, and some of which are directly involved in sialic acid binding (Weis et al., supra).
After receptor-mediated endocytosis into an endosomal compartment, HA undergoes a series low-pH-induced conformational changes to a fusogenic form which mediates fusion of the viral envelope with the host cell membrane, resulting in introduction of the core of the virus into the host cell.
Assumption of the membrane-fusion-promoting conformation by HA is dependent on low pH, and is not dependent on sialic acid-containing-receptor binding since low pH alone in the absence of sialic acid binding is able to render HA fusogenic. Thus, if the virus and host cell membranes are in close enough proximity, low pH is sufficient to trigger fusion. Indeed, in the presence of streptavidin, virosomes comprising HA and biotinylated lipids fuse in a low-pH dependent manner with liposomes which also comprise biotinylated lipids, but which do not comprise sialic acid (Schoen et al., 1996, FEBS Letters 390:315-318). Therefore, the sialic acid receptor binding most likely ensures that the two desired membranes are sufficiently close at the time the pH is lowered to enable fusion. Indeed, the physical proximity of the two membranes at the time of pH-induced HA conformational changes is crucial for successful membrane fusion, since for most HA types, low pH treatment in the absence of a target membrane results in irreversible fusion inactivation of HA.
The amino acid sequence and detailed structural information for HA have been reported (Wilson et al., 1981, Nature 289:366-373; Weis et al., 1988, Nature 333:426-431), as has been the sequence of the gene encoding HA (Verhoeyen et al., 1980, Nature 286:771-776). The sequence of this gene encoding HA is available in the GenBank (Accession No. V01085). The sequence of the influenza virus A strain X-31 containing the A/Aicha/2/6B (H3N2) gene is set forth in the GenBank at Accession No. J02090. Strain X-31 is the strain used to determine the crystal structure of HA, and strain X-31 was used in the experiments disclosed herein.
Although an enveloped virus preferentially incorporates its own viral envelope proteins into its envelope during viral packaging, the tropism of a number of enveloped viruses may be altered by the acquisition an envelope glycoprotein encoded by a different virus having a different tropism. The exogenous envelope protein is acquired during virus assembly by a process denoted phenotypic mixing or pseudotyping. Pseudotyped viruses can be formed by co-infection of a cell by two different enveloped viruses or they can be generated experimentally by expressing a nucleic acid encoding an exogenous viral envelope protein in a cell producing an enveloped virus. Pseudotype formation in vivo has been postulated to provide a mechanism whereby the pathologic potential of an enveloped virus can be modified by co-infection of host cells with a viruses encoding differing envelope proteins.
The production of a pseudotyped virus having an envelope fusion protein comprising a portion of a viral envelope protein fused with a portion of an exogenous protein recognized by a particular cell surface receptor was first reported by Kasahara et al. (1994, Science 226:1373-1376). Kasahara et al., replaced the amino-terminus of the ecotropic Murine Leukemia Virus (MLV) envelope protein Eco-Env with the polypeptide hormone erythropoietin (EPO) to form the fusion protein Eco-Env-EPO. Kasahara et al., demonstrated specific targeting of virions comprising Eco-Env-EPO to cells expressing the EPO cell surface receptor in tissue culture.
However, even in the presence of the wild type virus envelope, the infectious titers of the EPO-encoding pseudotyped MLV and of other similar retroviral vectors are generally too low to be useful in a clinical setting. In addition, alteration of viral envelope proteins for the purpose of altering the tropism of the virion has invariably affected the fusogenic capacity of the altered virion envelope protein to induce fusion of the virion envelope with the target cell membrane.
Thus, a significant unmet need remains for the development of an agent which can be incorporated into the envelope of a virion and which is capable of inducing fusion of the virion envelope with the membrane of a desired target cell, wherein the fusion-inducing capacity of the agent is substantially independent of the tropism of the virion. The present invention meets this need.
The invention relates to a lipid-containing vector capable of fusing to a cell membrane. The vector comprises a mutant hemagglutinin, wherein the hemagglutinin comprises a mutation in the receptor binding pocket of the hemagglutinin, wherein the mutation substantially abrogates binding of the hemagglutinin to a sialic acid containing receptor, and further wherein the mutation does not affect the fusogenic capacity of the hemagglutinin.
In one aspect, the hemagglutinin is an influenza A virus hemagglutinin.
In another aspect, the mutant hemagglutinin comprises a mutation in at least one amino acid in the receptor-binding pocket of the influenza A virus hemagglutinin.
In a preferred embodiment, the amino acid sequence of the mutant hemagglutinin differs from the amino acid sequence of wild type influenza A virus hemagglutinin in at least one of histidine-17, aspartic acid-112, threonine-115, glutamine-190, and leucine-226.
In another preferred embodiment, the amino acid sequence of the mutant hemagglutinin differs from the amino acid sequence of wild type influenza A virus hemagglutinin in at least one of histidine-17 and aspartic acid-112, and further in at least one of threonine-115, glutamine-190, and leucine-226.
In yet another preferred embodiment, the mutant hemagglutinin is selected from the group consisting of HA[T155S], HA[E190D], HA[L226V], HA[E190D,L226V], HA[T155S,L226V], HA[T155S,L226V,H17Q], HA[T155S,L226V,D112G], and HA[T155S,E190D].
In another aspect, the vector of the invention further comprises a targeting molecule.
In a preferred embodiment, the targeting molecule is selected from the group consisting of a viral envelope protein, an antibody, an antibody domain, an antigen, a T-cell receptor, a cell surface receptor, a cell surface adhesion molecule, a major-histocompatibility locus protein, a chimeric protein comprising at least a portion of Myc protein, a chimeric protein comprising at least a portion of Tva protein, a chimeric protein comprising at least a portion of EGF, and a peptide selected by phage display that binds specifically to a defined cell.
In another aspect, the vector of the invention comprises at least one additional component.
In a preferred embodiment, the additional component is selected from the group consisting of a nucleic acid, an antisense nucleic acid, a gene, a protein, a peptide, a Vpr protein, an enzyme, an intracellular antagonist of HIV, a radionuclide, a cytotoxic compound, an antiviral agent, and an imaging agent.
In another aspect, the vector of the invention is selected from the group consisting of an enveloped virus and a liposome.
Also included in the invention is a method of producing a lipid-containing vector. The method comprises pseudotyping an enveloped virus with a mutant influenza A virus hemagglutinin, wherein the mutant hemagglutinin comprises at least one amino acid substitution at residues threonine-115, glutamine-190, and leucine-226 in the receptor binding pocket of the hemagglutinin, and further wherein the substitution substantially abrogates binding of the hemagglutinin to a sialic acid containing receptor, and co-pseudotyping the virus with a targeting molecule.
In one aspect, the vector comprises an additional component.
In another aspect, the amino acid substitution is selected from the group consisting of a change from threonine to serine at residue 155, a change from glutamine to asparagine at residue 190, and a change from leucine to valine at residue 226.
In another aspect, the vector comprises an amino acid substitution from threonine to serine at residue 155, and a second amino acid substitution from leucine to valine at residue 226.
In yet a further aspect, the targeting molecule is selected from the group consisting of a viral envelope protein, an antibody, an antibody domain, an antigen, a T-cell receptor, a cell surface receptor, a cell surface adhesion molecule, a major histocompatibility locus protein, a chimeric protein comprising at least a portion of Myc protein, a chimeric protein comprising at least a portion of Tva protein, a chimeric protein comprising at least a portion of EGF, and a peptide selected by phage display that binds specifically to a defined cell.
In a preferred embodiment, the targeting molecule is a chimeric protein comprising at least a portion of Myc, at least a portion of Tva, and at least a portion of EGF.
In another preferred embodiment, the additional component is selected from the group consisting of a nucleic acid, an antisense nucleic acid, a gene, a protein, a peptide, a Vpr protein, an enzyme, an intracellular antagonist of HIV, a radionuclide, a cytotoxic compound, an antiviral agent, and an imaging agent.
The invention also includes an isolated nucleic acid encoding an influenza A virus hemagglutinin, wherein the nucleic acid comprises a mutation in the receptor binding pocket of the hemagglutinin, wherein the mutation substantially abrogates binding of the hemagglutinin to a sialic acid containing receptor, and further wherein the mutation does not affect the fusogenic capability of the hemagglutinin.
In one aspect, the mutation effects a conservative amino acid substitution.
In a preferred embodiment, the conservative amino acid is selected from the group consisting of threonine-155, glutamine-190, and leucine-226.
Also included is an isolated influenza A virus hemagglutinin wherein the hemagglutinin comprises a mutation which substantially abrogates binding of the hemagglutinin to a sialic acid containing receptor and further wherein the mutation does not affect the fusogenic capability of the hemagglutinin.
In addition, the invention includes a pseudotyped murine leukemia virus (MLV) comprising a mutant influenza A hemagglutinin, wherein the mutant hemagglutinin comprises a first mutation comprising a change from threonine to serine at amino acid 155, and further wherein the mutant hemagglutinin comprises a second mutation comprising a change from leucine to valine at amino acid 226, wherein the pseudotyped MLV expresses the mutant hemagglutinin and wherein the mutant hemagglutinin is in the envelope of the pseudotyped MLV.
In one aspect, the hemagglutinin further comprises a third mutation comprising an amino acid substitution which causes the mutant hemagglutinin to undergo low-pH induced conformational changes to a fusogenic form.
In a preferred embodiment, the third mutation comprises an amino acid substitution selected from the group consisting of a substitution from histidine to glutamine at amino acid 17 and a substitution from asparagine to glycine at amino acid 112.
The invention also includes a composition comprising a co-pseudotyped enveloped virus expressing a mutant hemagglutinin and a targeting molecule wherein the co-pseudotyped virus binds to a target cell expressing a receptor for the targeting molecule and further wherein the mutant hemagglutinin causes the virus to fuse with the target cell.
In addition, the invention relates to a mammalian cell comprising pseudotyped murine leukemia virus (MLV) comprising a mutant influenza A hemagglutinin, wherein the mutant hemagglutinin comprises a first mutation comprising a change from threonine to serine at amino acid 155, and further wherein the mutant hemagglutinin comprises a second mutation comprising a change from leucine to valine at amino acid 226, wherein the pseudotyped MLV expresses the mutant hemagglutinin and wherein the mutant hemagglutinin is in the envelope of the pseudotyped MLV.
The invention further relates to a mammalian cell comprising co-pseudotyped enveloped virus expressing a mutant hemagglutinin and a targeting molecule wherein the co-pseudotyped virus binds to a target cell expressing a receptor for the targeting molecule and further wherein the mutant hemagglutinin causes the virus to fuse with the target cell.
Also included in the invention is a method of targeting delivery of a component to a desired cell. The method comprises inserting a mutant, hemagglutinin and a targeting molecule on the surface of a vector, wherein the targeting molecule mediates binding of the vector to a targeting molecule-specific receptor on the cell, and further wherein the mutant hemagglutinin mediates membrane fusion of the vector with the membrane of the cell, thereby delivering the component to the cell.
In one aspect, the component is selected from the group consisting of a nucleic acid, an antisense nucleic acid, a gene, a protein, a peptide, a Vpr protein, an enzyme, an intracellular antagonist of HIV, a radionuclide, a cytotoxic compound, an antiviral agent, and an imaging agent.